JOURNAL OF CLINICAL MICROBIOLOGY, Dec. 1991, p. 2805-28080095-1137/91/122805-04$02.00/0Copyright © 1991, American Society for Microbiology

Vol. 29, No. 12

Sample Preparation Method for Polymerase Chain Reaction-BasedSemiquantitative Detection of Leptospira interrogarts Serovar

Hardjo Subtype Hardjobovis in Bovine UrineM. J. GERRITSEN,l* T. OLYHOEK,1 M. A. SMITS,2 AND B. A. BOKHOUT3

Departments of Bacteriology,' Molecular Biology,2 and Immunology,3 Central Veterinary Institute, P.O. Box 65,8200 AB Lelystad, The Netherlands, 03200-73253

Received 20 May 1991/Accepted 16 September 1991

An improved method of preparing bovine urine samples was developed for the rapid, specific, and sensitivedetection of Leptospira interrogans serovar hardjo (subtype hardjobovis) DNA by the polymerase chain reaction(PCR). A total of 100 leptospire-free cows, 4 experimentally infected cows, and 2 negative control cows wereused. PCR results were improved by (i) using 10-ml urine samples instead of 1-ml samples, (ii) adding 1 to108 Leptospira biflexa serovar patoc cells as a carrier to each treated sample, (iii) preventing the loss of pelletedleptospires, and (iv) minimizing the presence of PCR-inhibiting factors in the samples. The preparation methodenabled us to use the PCR to reproducibly detect as few as 5 to 10 leptospires per ml of urine without the needfor dot blot hybridization. In addition, we were able to estimate the number of leptospires shed by ex-perimentally infected cows.

Bovine leptospirosis is caused by Leptospira interrogansserovar hardjo subtype hardjobovis (referred to as subtypehardjobovis) and is classified as a zoonosis. Because theorganism persists in the kidneys and genitals without clinicalsigns of disease (6), carrier cows often excrete leptospires intheir urine. Such cattle are an important source of infection,not only for other cows, but also for dairy farm workers andother people (19).Although culture techniques can be used to detect lepto-

spires in urine, these techniques are slow and laborious. Forthat reason, many important aspects concerning subtypehardjobovis infections, for example, excretion patterns ofthe bacteria in bovine urine and the effectiveness of medi-cation, have been insufficiently investigated. The polymer-ase chain reaction (PCR) can rapidly and sensitively detectleptospires, but its use has been limited because no reliablesample preparation method has been available.

Various investigations have described PCR assays todetect microorganisms in urine (1, 7, 9, 13, 14), other bodilyfluids (5, 8, 10, 15, 16), or water (3). Van Eys et al. (17)described a sample preparation method to detect subtypehardjobovis DNA in bovine urine by PCR. This method wastested primarily on bovine urine samples collected sterilelyfrom the bladder. When this method was used to prepareurine samples collected in the field, the detection of subtypehardjobovis DNA by PCR was not reproducible.

In the present study, we developed an improved methodfor preparing bovine urine samples collected nonsterilelyfrom the bladder and evaluated its use in the PCR fordetecting leptospiral DNA.

MATERIALS AND METHODS

Bacterial strains and growth conditions. Table 1 lists thebacterial strains used in this study. The leptospiral strains L.interrogans serovar hardjo subtype hardjobovis and L. bi-flexa serovar patoc were cultured in EMJH medium (10) at29°C. The medium in which subtype hardjobovis was cul-

* Corresponding author.

tured was enriched with 5% rabbit serum. The nonleptospi-ral strains were plated onto blood agar plates and incubatedat 37°C for 24 h.

Subtype hardjobovis from the urine of experimentallyinfected cows was cultured by the method described byKuiken et al. (12).

Urine samples. Urine samples were collected from 100cows that were serologically negative for subtype hardjobo-vis and housed on two different farms where subtype hard-jobovis was not present, 4 cows that were experimentallyinfected with subtype hardjobovis, and 2 cows that wereused as negative controls. A 5-ml amount of the diureticDimazon (Hoechst) was administered intravenously to eachcow. About 10 min later, after the vulva of each cow wascleaned with tap water so that feces would not contaminatethe urine, a 100-ml midstream sample of urine was collected.

Specificity of the primers. The primers used in the PCR testwere described by Van Eys et al. (17). In the present study,their specificity was tested by subjecting 12 nonleptospiralmicroorganisms that may be contained in cattle urine (18), L.biflexa serovar patoc, and three subtype hardjobovis strainsto PCR (Table 1). Individual colonies of the 12 nonleptospiralmicroorganisms were selected and suspended in 1 ml ofbovine urine containing no leptospires to a cell density of>103/ml. The leptospiral strains were suspended likewise andto the same cell density. The cells were washed once with 1mM EDTA (pH 8.0) and once with sterilized water, and theDNA was then recovered as described below. An amount of10 ,ul of the resulting extract was subjected to PCR.Recovery of DNA. A 40-ml amount of each urine sample

was poured into a 50-ml tube containing 10 ml of 0.1 MEDTA (pH 8.0) and 0.5% formaldehyde (Merck). A 150-pulvolume of an L. biflexa serovar patoc culture (containing aminimum of 107 to 108 cells per ml of medium) was added toa 10-ml urine mixture. Another 10 ml of the same urinesample was seeded with either 5 or 50 subtype hardjoboviscells per ml (positive control) and 150 pul of an L. biflexaserovar patoc culture. The 10-ml samples were centrifugedat 13,000 x g for 20 min, after which 9 ml of supernatant wasremoved. The pellet was resuspended in the remaining

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2806 GERRITSEN ET AL.

TABLE 1. Specificity of the PCR assay

Microorganism DbeyeCRn

Acinetobacter calcoaceticus........................................Alcaligenes faecalis ...................................................Actinomyces pyogenes...............................................Corynebacterium renale .............................................Enterobacter cloacae.................................................Escherichia coli........................................................Klebsiella species......................................................Leptospira biflexa serovar patoc ..................................Leptospira interrogans serovar hardjo subtype

hardjobovis Sponselee............................................. +Leptospira interrogans serovar hardjo subtype

hardjobovis Mulder ................................................ +Leptospira interrogans serovar hardjo subtype

hardjobovis 146...................................................... +Proteus mirabilis.......................................................Proteus vulgaris........................................................Pseudomonas aeruginosa ...........................................Staphylococcus aureus (hemolytic)...............................Streptococcus faecalis ...............................................

solution and transferred into a 1-ml tube with a screw cap(Starstedt). The resulting 1-ml suspension was centrifugedfor 10 min at 13,000 x g, after which the supernatant wasremoved by a glass pipette, leaving behind about 100 il1 offluid. The pellet in this fluid was washed twice, once with 1ml of 1 mM EDTA (pH 8.0) and once with 1 ml of water. Inboth cases, the 1-ml suspension was centrifuged for 10 min at13,000 x g, after which the supernatant was removed,leaving behind about 100 tl of fluid. The final remaining 100jxl was vortexed, and the DNA was released by incubatingthe samples for 10 min at 100°C. The samples were theneither stored at -20°C or subjected to PCR.PCR. A 25-pul amount of each prepared urine sample was

subjected to PCR in a total volume of 50 pl. The reactionmixture contained 10 mM Tris hydrochloride (pH 9.0), 50mM KCl, 2 mM MgCl2, 0.1 mM each of the four deoxynu-cleotide triphosphates (Pharmacia), 100 pM primers (16), 0.5U of Taq DNA polymerase (Perkin-Elmer, Cetus), and0.01% (wt/vol) gelatin. The mixtures were covered with 50 pulof mineral où, placed in an automatic PCR processor (Ce-tus), and subjected to 36 5-min cycles, each consisting ofdenaturation for 1 min at 94°C, annealing of primers for 1 minat 55°C, and extension for 3 min at 72°C. A final 37th cyclewas identical, except that the extension step lasted 10 mininstead of 3 min. PCR products were analyzed by gelelectrophoresis (17).

Sensitivity and semiquantification. A Hawksley-Englandcounting chamber was used to count subtype hardjobovis cells.

Serial twofold dilutions of subtype hardjobovis cells weremade in 10 ml of subtype hardjobovis-free urine (100, 50, 25,10, 5, and 0 cells per ml). DNA was recovered as describedabove, and 25 pul of the resulting extract was subjected to PCR.For the quantification of leptospires in urine samples, 10-pul

samples were diluted in 40 of water after DNA wasrecovered as described above. From this point on, serialtwofold dilutions were made by diluting 25 pul of each dilutionin 25 pil of water. The total volume was subjected to PCR.

Experimentally induced infections. All cattle were intraoc-ularly inoculated in each eye with 0.5 ml of the bacterialculture (no less than 107 to 108 cells per ml) on fourconsecutive days. Two cows (no. 789 and no. 790) wereinfected with virulent L. interrogans serovar hardjo subtype

10 50 25 10 5 0 p w

240 bp,. _

250 12563 25 12 0

FIG. 1. Gel electrophoresis of PCR products obtained from urinesamples seeded with 100, 50, 25, 10, 5, and 0 subtype hardjobovis cellsper ml. The numbers above the lanes indicate the concentrations ofsubtype hardjobovis cells in the urine samples. The numbers of sub-type hardjobovis in the reaction mixtures are indicated under the lanes.The 240-bp DNA fragment that was amplified is indicated by an arrow.

hardjobovis 146 (kindly provided by B. Ellis); two cows (no.791 and no. 792) were infected with virulent subtype hard-jobovis Mulder (kindly provided by Z. Bercovich). Cattleinfected with the same virulent strain were placed together inan isolated cubicle. Two cows (no. 793 and no. 794) wereused as negative controls. They were placed in an isolatedcubicle and inoculated in the same way with EMJH medium.Blood samples (10 ml) and urine samples (100 ml) were

collected twice weekly from each cow for 3 months. Theblood samples were tested by the enzyme-linked immuno-sorbent assay (ELISA) as described earlier (2) and by themicroscopic agglutination test. The urine samples werecollected and assayed by PCR for the presence of subtypehardjobovis DNA as described above.

RESULTS

Specificity of the PCR assay. The specificity of the primerset used in the PCR was tested with microorganisms thatmight be contained in bovine urine. The PCR only amplifiedDNA from the three subtype hardjobovis strains (Table 1).

Positive control. To detect false-negative results, we intro-duced a positive control for each urine sample. Each urinesample was divided into two 10-ml portions and one wasroutinely seeded with 50 subtype hardjobovis cells per ml.All positive control samples from all tested urine samples(about 300) reacted positively. These findings suggested thatthe DNA of 50 subtype hardjobovis cells per ml of urinecould reproducibly be amplified by PCR when our samplepreparation method was used.

Evaluation of the sample preparation method. By using theimproved sample preparation method, we obtained ethidiumbromide-stained amplified DNA fragments on an agarose gelfrom urine samples containing as few as 5 to 10 subtypehardjobovis cells per ml of urine (Fig. 1).

Results of the PCR were considered reliable because ofthe following: (i) all experimentally infected animals becamePCR positive, which was confirmed by culturing; (ii) bothnegative control animals remained PCR negative during thewhole observation period; (iii) all positive control urineswere always PCR positive (Table 2); and (iv) we were able toobtain identical results from collected urine samples testedin duplicate (results not shown).To test whether the reliability of the PCR assay was influ-

enced by variations in individual urine samples, we tested 100different samples collected under field conditions from cowsthat were free of subtype hardjobovis. All positive controlsamples from these cows reacted positively in the PCR(results not shown), indicating that differences in urine com-

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SEMIQUANTITATIVE DETECTION OF SUBTYPE HARDJOBOVIS 2807

TABLE 2. Results of serologic tests and PCR from theexperimentally infected animals during the observation period

Days after No. of positive cowsainfection MAT ELISA PCRb

o o o o4 0 0 07 0 0 0

il 4 1 014 4 1 018 4 2 421c 4 2 425 4 2 428 4 3 432 4 3 435 4 3 439 4 3 442-81 4 4 484 4 4 388 4 4 391 4 4 295 4 4 2

a Six cows were used in each test. Two of the cows were used as negativecontrols; their serum and urine samples remained negative in serologic testsand in the PCR, respectively. MAT, microscopic agglutination test.

b Ail urine samples seeded with 5 or 50 subtype hardjobovis cells per mlwere positive in the PCR.

C Ail urine samples of the experimentally infected cows were also positivewhen cultured.

789 790 791- C - C ; -1 ~er w v r:,%

792 793 794,a 1- -1 C- - 1r-

FIG. 2. Gel electrophoresis of PCR products of bovine urinesamples collected 91 days after infection from experimentally infectedcows, no. 789, 790, 791, and 792 as well as the two negative controlcows, no. 793 and 794. Abbreviations: cl., urine samples withoutleptospires added; +50, urine samples seeded with 50 subtypehardjobovis cells per ml of urine; +5, urine samples seeded with 5subtype hardjobovis cells per ml of urine; P, leptospire-free urinesample seeded with L. biflexa serovar patoc; W, washing solutions.

subtype hardjobovis cells per ml of urine. After that time, cowno. 789 shed fewer leptospires, but cow no. 790 continued toshed about 1 x 104 to 4 x 104 Leptospira cells per ml of urine.Most samples show an ethidium bromide-stained band

below the specific subtype hardjobovis band (240 bp) on a gel(Fig. 2 and 3). This lower band represents unused primers,and thus the intensity of this band varied among the samples.

DISCUSSION

position did not influence the detection of subtype hardjobo-v1s.

In addition, blood and urine samples were collected fromexperimentally infected cows (Table 2). Before infection,these samples were serologically and PCR negative. At 18days after infection, however, all blood and urine sampleswere serologically and PCR positive. At 21 days afterinfection, the results of the PCR were confirmed by culturingthese urine samples so that false-positive results were ex-cluded. Although samples from two cows (no. 791 and no.789) were PCR negative but serologically positive at day 84and 91, respectively, false-negative PCR results were ex-cluded because the positive controls of these samples (seed-ed with 5 or 50 subtype hardjobovis cells per ml) reactedpositively in the PCR (Fig. 2). These findings indicate thatthese samples contained fewer than 5 subtype hardjoboviscells per ml of urine or none at all. Subtype hardjobovisDNA was detected in urine samples for 66 days or longer.The two negative control cows remained negative during thewhole observation period.

Evaluation of a semiquantitative method to detect lepto-spires. Urine samples were seeded with various amounts ofleptospires to determine the accuracy of the semiquantita-tive method (results not shown). There was a direct corre-lation between the intensity of the ethidium bromide-stainedDNA bands and the number of leptospires contained in thefinal reaction mixture (Fig. 1). The ethidium bromide-stainedband with the lowest intensity represented amplified DNAfragments derived from the DNA of 10 to 20 organismscontained in the PCR mixture (Fig. 1). In addition to theseeded urine samples, eight urine samples collected fromtwo experimentally infected cows (no. 789 and no. 790) werealso examined by the semiquantitative method (Fig. 3). Thenumbers of subtype hardjobovis cells shed by both cowsincreased during the infection. The shedding of subtypehardjobovis peaked on day 53 after infection at 4 x 104

Although the PCR can rapidly and specifically detect DNAeven in very small numbers of microorganisms, its use fordetecting leptospires in bovine urine samples has beenlimited because of the lack of a proper method for preparingthe samples.We improved the urine sample preparation method de-

scribed by Van Eys et al. (17) in four ways, so that when thesamples were tested by PCR, results were specific andreliable even from urine samples containing as few as 5 to 10subtype hardjobovis cells per ml of urine. First, the volume ofthe sample was increased from 1 to 10 ml, which greatlyenhanced the reproducibility of our results, especially withurine samples containing only small numbers of bacterialcells. Second, the quantitative sedimentation of leptospireswas enhanced by adding 107 to 108 L. biflexa serovar patoc

days after infectionno. cow

789 PCRquant.

7 21 35 45 53 9 77 94

''l'il'lo 10 10 21 ' 41U' 10' 210 0

1101 11 111790 D(, --

cuant. 3O 60; 510 5 10 4 ('0 1 0 4 '.;

FIG. 3. Quantification (quant.) of subtype hardjobovis shed bycow no. 789 and no. 790 on days 7, 21, 35, 45, 53, 63, 77, and 91 afterinfection, compared with the intensities of the ethidium bromide-stained bands obtained after the amplification of 25 ,ul of preparedurine samples and gel electrophoresis (PCR) on the same days.

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2808 GERRITSEN ET AL.

cells as a carrier to facilitate the collection of leptospiral cellsfrom urine samples. Third, the loss of leptospiral cells duringpreparation was prevented by allowing a certain amount offluid to remain above the pellet after each centrifugation step.

Finally, we minimized the presence of PCR-inhibitingfactors in the samples in three ways. First, we attempted tocollect urine samples that were as clean as possible (seeMaterials and Methods). Van Eys et al. (17) mainly collectedsterile urine samples from the bladder, but we found thismethod impractical and, more importantly, unsuitable forstudying numerous cows. Instead, we used a diuretic, Dim-azon, which causes no injuries (which can occur duringcatheterization) and is easy to use routinely in the field.Second, EDTA was added to the urine sample to preventDNase activity during the storage and transport of thesamples. Because EDTA was removed during the washingprocedures, it did not inhibit the PCR. Finally, leptospiralcells were washed twice to eliminate small amounts ofinhibiting factors still contained in the urine (see Materialsand Methods). Although other techniques can effectivelyeliminate PCR-inhibiting factors, for example, phenol-chlo-roform extraction (7), the use of Geneclean (17), and the useof silica particles or diatoms (4), they can allow smallamounts of DNA to be lost and thus decrease the sensitivityand reproducibility of the PCR results. These techniqueshave the additional drawback of being more laborious thanthe method described in this article and often involve the useof noxious chemicals.

Because culturing leptospires is laborious, slow (at least 6months must elapse before a sample can be confirmed to beleptospire negative), and probably less sensitive than thePCR, we introduced a positive control to detect false-negativeresults and to be able to exclude culturing (see Results).Because the method was less sensitive in the beginning of theexperiment, we routinely used 50 subtype hardjobovis cellsper ml of urine as a positive control instead of the 5 subtypehardjobovis cells per ml of urine which can be detected now.

Urine samples stored as long as 1 month at 4°C inEDTA-formaldehyde continued to yield positive results.Storing samples at -20°C and freeze-thawing them had noeffect on the amplification of DNA. These are considerableimprovements over the culturing technique, which must beperformed immediately after samples are collected.

Leptospires shed by experimentally infected cattle werealso reproducibly detected. Furthermore, we were able toestimate for the first time the concentration of subtypehardjobovis in the urine samples. Because the concentrationof leptospires in urine is probably volume dependent, vari-ations in diuresis should be considered.Because of the enormous quantities of subtype hardjobo-

vis shed by infected cattle, future research must focus onhow to prevent shedding. The experimentally induced infec-tions suggest that the serologic response is correlated withthe amount of Leptospira cells shed. This possibility shouldbe verified by testing a larger number of cattle.The sample preparation method described in this article

enables us to use the PCR to reliably detect subtype hard-jobovis in bovine urine. This rapid, sensitive, and specificassay paves the way for studying patterns in shedding andassessing the efficacy of vaccination or antibiotics in thetreatment of bovine leptospirosis.

ACKNOWLEDGMENTSThis work was supported by grants from the Veterinary Depart-

ment of the Ministry of Health and from the Veterinary Service of theDutch Ministry of Agriculture, Nature, Management, and Fisheries.

We thank B. Meeuwissen for performing the serological tests, A.Lammers for practical assistance, A. Moerman and B. E. C.Schreuder for their help in collecting the urine and blood samples,W. J. Terpstra for providing the primers, and P. J. van der Heydenand P. Schluck for their helpful criticism of the article.

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